Dna sequence for horseradish peroxidase c modified for expression in mammalian cells

ABSTRACT

Synthetic DNA coding for horseradish peroxidase includes the following sequence: ##STR1## and incorporates useful restriction sites at frequent intervals to facilitate the cassette mutagenesis of selected regions. Also included are flanking restriction sites to simplify the incorporation of the gene into any desired expression system.

This invention relates to synthetic genes coding for horseradishperoxidase.

Horseradish peroxidase C (E.C.1.11.1.7) (HRP) is the major peroxidaseisozyme isolated from the horseradish (Armoracia rusticana). It is amonomeric glycoprotein of 308 amino acids the polypeptide chain having aMW of 33,980 D. There are three neutral carbohydrate side chains and 4disulphide bridges. The amino acid sequence of the mature protein hasbeen determined. The presence of a pyrrolidonecarboxylyl amino terminusindicates that the protein is probably produced as a precursor form thatis processed on secretion. The active form of the enzyme contains ahemin prosthetic group.

The enzyme is particularly stable and is amenable to crosslinking andderivitisation without excessive loss of activity. This together withits wide range of chromogenic substrates, some of which give rise toinsoluble, chemiluminescent or flourescent products, and the lowbackground activities observed in most applications, have madehorseradish peroxidase an invaluable tool for diagnostic and researchapplications in the fields of immunology, histochemistry, cytology andmolecular biology. A further advantage it presents over other enzymaticmarkers is that some some substrates for the enzyme give rise toelectron dense products that allow correlation of peroxidase locationwith cellular ultrastructure using electron microscopy. In addition,horseradish peroxidase is electron dense itself by virtue of the Fe itcontains and as a result can act as an E.M. marker in its own right.Particular applications have been found in immunochemistry, whereperoxidase cross linked to immunoglobulin is widely used in both ELISAbased assay systems and immunocytochemistry. Methods have been describedthat use either direct crosslinking of peroxidase to the immunoglobulinor indirect crosslinking of biotin labelled immunoglobulin to astreptavidin/horseradish peroxidase complex. Such streptavidin complexeshave also found widespread application in nucleic acid hybridisationmethods where biotinylated probe sequences can be localised bysequential incubation with the streptavidin/peroxidase complex and asuitable chromogenic peroxidase substrate.

The amino acid sequence of horseradish peroxidase is taught by Welinder,K. G. (Eur. J. Biochem. 96, 483-502 (1979)). The cloning of the cDNA ornatural gene for horseradish peroxidase has not been described.

In order to facilitate the dissection of the structure/functionrelationships of HRP, its incorporation into expression vectors and theproduction of novel chimeric proteins containing HRP functionality animproved novel synthetic gene for the peroxidase C produced by Armoraciarusticana is sought.

It is by no means easy to predict the design of an improved HRP gene,since the factors that determine the expressibility of a given DNAsequence are still poorly understood. Furthermore, the utility of thegene in various applications will be influenced by such considerationsas codon usage and restriction sites. The present invention relates to asynthetic HRP gene which has advantages in the ease with which it can bemodified due to the presence of useful restriction sites.

When synthesising and assembling genes, problems have been encounteredwhen there are inverted or direct repeats greater than eight bases longin the genetic sequence. In addition, areas of unbalanced basecomposition such as G/C or A/T rich regions or polypurine/polypyrimidinetracts have been found to lead to inefficient expression. :The presentinvention seeks to overcome or at least alleviate these difficulties.

According to a first aspect of the invention, there is provided DNAcoding for HRP and having restriction sites for the following enzymes:##STR2## The DNA may also contain a 5' HindIII site and/or a 5' NdeIsite and/or a 3' BamHI site and/or a 3' EcoRI site.

According to a second aspect of the invention, there is provided DNAincluding the following sequence: ##STR3## The above sequence may beimmediately preceeded by an initiation codon (ATG) and immediatelyfollowed by a termination codon (TAA), but this will not necessarily bethe case if the DNA incorporates linker(s) and/or extension(s), such asa sequence coding for a signal peptide, for example for efficientexpression in eukaryotic cells such as mammalian cells. One extensionwhich gives good expression in mammalian cells is a 5' -extension codingfor the amino acids KCSWVIFFLMAVVTGVNS, which may be provided between aninitiation codon and the codon coding for the first Q residue. Apreferred such extension is shown in FIG. 6. A sequence coding for a3'-signal sequence may code for LLHDMVEVVDFVSSM; a preferred DNAsequence coding for this series of amino acid residues is also shown inFIG. 6.

A synthetic HRP gene as described above incorporates useful restrictionsites at frequent intervals to facilitate the cassette mutagenesis ofselected regions. Also included in preferred embodiments are flankingrestriction sites to simplify the incorporation of the gene into anydesired expression system.

Codons are those that are favoured by E. coli but it is expected thatthe DNA would be suitable for expression in other organisms includingyeast and mammalian cells.

According to a third aspect of the invention, there is provided agenetic construct comprising DNA according to the first or second aspector a fragment thereof. The fragment may comprise at least 10, 20, 30, 40or 50 nucleotides. A genetic construct in accordance with the thirdaspect may be a vector, such as a plasmid, cosmid or phage.

According to a fourth aspect of the invention, there is provided aprocess for the preparation of DNA in accordance with the first orsecond aspect or a genetic construct in accordance with the thirdaspect, the process comprising coupling successive nucleotides and/orligating appropriate oligomers.

The invention also relates to other nucleic acid (including RNA) eithercorresponding to or complementary to DNA in accordance with the first orsecond aspects.

The invention encompasses a process for the production of monodispersehorseradish peroxidase C comprising the expression of at least part of agenetic construct as described above.

Further, the invention extends to constructs as described abovecomprising all or a fragment of a sequence in accordance with the firstor second aspect fused to any other sequence of DNA so as to result in asequence capable of encoding a hybrid protein possessing peroxidaseactivity. An example of such a construct is a genetic fusion between agene encoding horseradish peroxidase and a gene encoding streptavidin oravidin such that the encoded fusion protein possesses both biotinbinding and peroxidase activity. Another example is a genetic fusionbetween a gene encoding horseradish peroxidase and a gene encoding animmunoglobulin-derived antigen binding function such that the fusionprotein possesses both antigen binding and horseradish peroxidaseactivity. The antigen binding function may be an immunoglobulin heavychain or light chain or fragments thereof or an engineered monomericantigenic recognition site.

Particular constructs of interest include: vectors comprising the genefor horseradish peroxidase C that enable the production of fusionsbetween horseradish peroxidase and any other protein of interest; andexpression vectors that provide for the co-expression of the gene forhorseradish peroxidase and another gene of interest either as a singlefusion product, as a single polycistronic message or as two separate butlinked transcriptional units.

According to a further aspect of the invention, there is provided a genefor horseradish peroxidase containing a mutation (either missense,nonsense, deletion, insertion, duplication or other rearrangement) thatdestroys or impairs the activity of the encoded horseradish peroxidaseprotein. The invention extends to genetic constructs including all or afragment of such a mutant horseradish peroxidase gene.

Defective or non-defective horseradish peroxidase genetic constructascan be employed (for example as markers) in mammalian cells and/or intransgenic animals.

Specific applications of synthetic genes for horseradish peroxidase,which themselves form further aspects of the invention, are disclosed ingreater detail below:

1) The gene can be incorporated into a suitable expression vector toallow for the efficient production of the enzyme in a compatibleorganism. This will have the advantage of being a ready source of amonodisperse enzyme preparation free of the contaminating isozymespresent in the material isolated from horseradish root. Varying theorganism or cell type chosen for production will also allow for theproduction of HRP with different patterns of glycosylation, including noglycosylation. Such material will have better defined properties thatwill make it more suitable for more demanding histochemical applicationsand sensitive enzyme assays, especially immunoassays.

2) The gene can be incorporated into an HRP-streptavidin or HRP-avidingene fusion. This will allow for the production of streptavidin-HRP oravidin-HRP complexes without the need for cross-linking. Again this willallow for a better defined, more stable product and will probably resultin less loss of both biotin binding and peroxidase activity.

3) Similarly, fusions between immunoglobulins and HRP or protein A andHRP can be produced that would be valuable histochemical reagents. Againthe need for the usual cross-linking procedures would be avoided.

4) The HRP gene would have valuable applications in the construction ofvectors designed to allow the production of fusions between HRP and anyother protein for which a gene or cDNA had been cloned or for which theamino acid sequence is known. This would be useful both for monitoringthe expression of a gene the product of which is difficult to assay andto tag the protein of interest to allow its metabolism andpharmodynamics to be followed in vivo by the use of the appropriatehistochemical techniques or enzyme assays. Additionally, HRP fusionswill allow for a simple immunopurification of the fusion product throughthe use of an appropriate anti-HRP antibody.

5) The expression of HRP will be a useful marker in expression systems,e.g. mammalian cell expression systems. The HRP gene could be expressedeither as a fusion or on a polycistronic message with the gene ofinterest, or as a separate but closely linked transcriptional unit. Theproduction of the easily assayed HRP could be readily screened for andused as an indication as to which clones of cells were likely to beexpressing large quantities of the desired product. The use offluorescent or chemiluminescent HRP chromogenic substrates would allowfor the possibility of directly selecting high producing eukaryoticcells by fluorescence activated cell-sorting (FACS).

6) HRP genes carrying mutations (missense, nonsense, deletion,insertion, duplication or other rearrangement) that destroy or impairthe enzymatic activity of the resultant product would allow theconstruction of vectors that could be used to follow the frequency ofreversion or suppression of the particular mutation introduced into thegene.

The introduction of such defective HRP genes into the germ line of theorganism of interest would also enable a researcher to fate-mapparticular cell-types by histologically examining the pattern of HRPactivity in the tissue of interest. Care would have to be excercised inconstructing a mutant HRP gene with the correct in vivo reversion rateso that areas of HRP activity and hence the presence of reverted HRPgene could be taken as evidence for the clonal origin of the HRP+ cells.The intact synthetic non-mutant gene could also be used for suchfate-mapping experiments by infection of an organism with the HRP genein a siutable vector such as a retroviral vector or transposon.

7) The advantage of a synthetic gene for HRP allows for the productionof HRP genes modified to encode a protein carrying small additionalsequences, such as N- or C- terminal extensions. These will be of greatapplication in simplifying the purification of the HRP and/or increasingthe ease and enhancing the specificity with which it can be cross-linkedto other proteins of interest or otherwise derivatised. For example, aC-terminal extension of six to eight Arg residues could be used tosimplify purification by analogy with the technique of Sassenfeld et al.Bio/technology 2 76 (1984). Alternatively, a tail of Lys residues wouldprovide an accessible and sensitive site for reaction with bifunctionalcross-linking reagents such as glutaraldehyde.

Preferred embodiments and examples of the invention will now bedescribed. In the following description, reference is made to a numberof drawings, in which:

FIG. 1, shows the amino acid sequence of horseradish peroxidase C;

FIGS. 2a-d shows the sequence of the horseradish peroxidase syntheticgene; a summary of useful restriction sites; and a sequence of front andback halves of the gene that were initially cloned;

FIGS. 3a-b shows a sequence of synthetic horseradish peroxidase genedivided into oligonucleotides;

FIGS. 4a-c shows a summary of assembly procedure used;

FIG. 5 shows the structure of the HRP E. coli expression plasmid pSD18;

FIGS. 6a-c shows a synthetic HRP gene modified for efficient expressionin mammalian cells; and

FIG. 7 shows the structure of the HRP mammalian expression plasmidpCP21.

EXAMPLE 1

The gene was designed to be synthesised and cloned, in this example, intwo halves with a final sub-cloning step to yield the full length gene.The sequence of the two halves of the gene together with that of thefinal product are depicted in FIG. 2. The final synthetic gene encodesthe entire mature horseradish peroxidase protein together with therequired initiator methionine residue but lacks the leader sequence thatis assumed to be present in the natural gene. It is envisaged that theleader sequence appropriate to the expression system of choice would beadded to the synthetic gene as required or ommitted to allow forintracellular expression of the gene.

The desired gene sequence was divided into a front half and a back halfof 501 and 474 bp respectively. Both halves were designed with a commonXhoI site to allow for the complete gene to be assembled with a simplecloning step. The front and back halves of the gene were divided into 24and 22 oligodeoxyribonucleotides (oligomers) respectively as depicted inFIG. 3. The division was such as to provide 7 base cohesive ends afterannealing complementary pairs of oligomers. The end points of theoligomers were chosen to minimise the potential for inappropriateligation of oligomers at the assembly stage.

The oligomers were synthesised by automated solid phase phosphoramiditechemistry. Following de-blocking and removal from the controlled poreglass support the oligomers were purified on denaturing polyacrylamidegels, further purified by ethanol precipitation and finally dissolved inwater prior to estimation of their concentration.

All the oligomers with the exception of the 5' terminal oligomers BB279and BB302 for the front half and BB303 and BB324 for the back half werethen kinased to provide them with a 5' phosphate as required for theligation step. Complementary oligomers were then annealed and theoligomers ligated together by T4 DNA ligase as depicted in FIG. 4. Theligation products were separated on a 2% low gelling temperature (LGT)gel and the bands corresponding to the front and back halves of thehorseradish peroxidase gene were cut out and extracted from the gel. Thepurified fragments were then ligated separately to EcoRI/HindIII cut DNAof the plasmid vector pUC18. The ligated products were transformed intoHW87 and plated on L-agar plates containing 100 mcg ml⁻¹ ampicillin.Colonies containing potential clones were then grown up in L-brothcontaining ampicillin at 100 mcg ml⁻¹ and plasmid DNA isolated. Positiveclones were identified by direct dideoxy sequence analysis of theplasmid DNA using the 17 base universal primer, a reverse sequencingprimer complementary to the opposite strand on the other side of thepolylinker and some of the oligomers employed in the assembly of thegene that served as internal primers. One front half and one back halfclone were subsequently re-sequenced on both strands to confirm that nomutations were present. The complete gene was then assembled byisolating the 466 bp XhoI-EcoRI fragment from the back half calone thatcontained the 3' end of the gene and ligating it to a front half clonethat had also been digested with EcoRI and XhoI. The identity of thefinal construct was confirmed by restriction analysis and subsequentcomplete resequencing.

All the techniques of genetic manipulation used in the manufacture ofthis gene are well known to those skilled in the art of geneticengineering. A description of most of the techniques can be found in oneof the following laboratory manuals: Molecular Cloning by T. Maniatis,E. F. Fritsch and J. Sambrook published by Cold Spring HarborLaboratory, Box 100, New York, USA, or Basic Methods in MolecularBiology by L. G. Davis, M. D. Dibner and J. F. Battey published byElsevier Science publishing Co. Inc. New York, USA.

Additional and modified methodologies are detailed below.

1) Oligonucleotide Synthesis

The oligonuoleotides were synthesised by automated phosphoramiditechemistry using cyanoethyl phosphoramidtes. The methodology is nowwidely used and has been described (Beaucage, S. L. and Caruthers, M. H.Tetrahedron Letters. 24, 245 (1981)).

2) Purification of Oligonucleotides

The oligonucleotides were de-protected and removed from the CPG supportby incubation in concentrated NH3. Typically, 50 mg of CPG carrying 1micromole of oligonucleotide was de-protected by incubation for 5 hr at70° in 600 mcl of concentrated NH₃. The supernatant was transferred to afresh tube and the oligomer precipitated with 3 volumes of ethanol.Following oentrifugation the pellet was dried and resuspended in 1 ml ofwater. The concentration of crude oligomer was then determined bymeasuring the absorbance at 260 nm.

For gel purification 10 absorbance units of the crude oligonucleotidewere dried down and resuspended in 15 mcl of marker dye (90% de-ionisedformamide, 10 mM tris, 10 mM borate, 1 mM EDTA, 0.1% bromophenol blue).The samples were heated at 90° for 1 minute and then loaded onto a 1.2mm thick denaturing polyacrylamide gel with 1.6 mm wide slots. The gelwas prepared from a stock of 15% acrylamide, 0.6% bisacrylamide and 7Murea in 1 X TBE and was polymerised with 0.1% ammonium persulphate and0.025% TEMED. The gel was pre-run for 1 hr. The samples were run at 1500V for 4-5 hr. The bands were visualised by UV shadowing and thosecorresponding to the full length product cut out and transferred tomicro-testubes. The oligomers were eluted from the gel slice by soakingin AGEB (0.5M ammonium acetate, 0.01M magnesium acetate and 0.1% SDS)overnight. The AGEB buffer was then transferred to fresh tubes and theoligomer precipitated with three volumes of ethanol at -70° for 15 min.The precipitate was collected by centrifugation in an Eppendorfmicrofuge for 10 min, the pellet washed in 80% ethanol, the purifiedoligomer dried, redissolved in 1 ml of water and finally filteredthrough a 0.45 micron micro-filter. The concentration of purifiedproduct was measured by determining its absorbance at 260 nm.

3) Kinasing of Oligomers

250 pmole of oligomer was dried down and resuspended in 20 mcl kinasebuffer (70 mM Tris pH 7.6, 10 mM MgCl2, 1 mM ATP, 0.2 mM spermidine, 0.5mM dithiothreitol). 10 u of T4 polynucleotide kinase was added and themixture incubated at 37° for 30 min. The kinase was then inactivated byheating at 85° for 15 min.

4) Annealing

8 mcl of each oligomer was mixed, heated to 90° and then slow cooled toroom temperature over a period of an hour.

5) Ligation

5 mcl of each annealed pair of oligomers were mixed and 10 X ligasebuffer added to give a final ligase reaction mixture (50 mM Tris pH 7.5,10 mM MgCl₂, 20 mM dithiothreitol, 1 mM ATP. T4 DNA ligase was added ata rate of 100 u per 50 mcl reaction and ligation carried out at 15° for4 hr.

6) Agarose Gel Electrophoresis

Ligation products were separated using 2% low gelling temperatureagarose gels in 1 X TBE buffer (0.094M Tris pH8.3, 0.089M boric acid,0.25 mM EDTA) containing 0.5 mcg ml⁻¹ ethidium bromide.

7) Isolation of Ligation Products

The band corresponding to the expected horseradish peroxidase gene orgene fragment ligation product was identified by reference to sizemarkers under long wave UV illumination. The band was cut out of the geland the DNA extracted as follows.

The volume of the gel slice was estimated from its weight and thenmelted by incubation at 65° for 10 min. The volume of the slice was thenmade up to 400 mcl with TE (10 mM Tris pH 8.0, 1 mM EDTA) and Na acetateadded to a final concentration of 0.3M. 10 mcg of yeast tRNA was alsoadded as a carrier. The DNA was then subjected to three rounds ofextraction with equal volumes of TE equilibrated phenol followed bythree extractions with ether that had been saturated with water. The DNAwas precipitated with 2 volumes of ethanol, centrifuged for 10 min in amicrofuge, the pellet washed in 70% ethanol and finally dried down. TheDNA was taken up in 20 mcl of TE and 2 mcl run on a 2% agarose gel toestimate the recovery of DNA.

8) Cloning of Fragments

For the initial cloning of the two halves of horseradish peroxidase 0.5mcg of pUC18 DNA was prepared by cleavage with HindIII and EcoRI asadvised by the suppliers. The digested DNA was run on an 0.8% LGT geland the vector band purified as described above. For the final assemblystep the clone carrying the front half of the horseradish peroxidasegene was treated similarly using the enzymes XhoI and EcoRI.

20 ng of cut vector DNA was then ligated to various peroxidase gene DNAranging from 2 to 20 ng for 4 hr using the ligation buffer describedabove. The ligation products were used to transform competent HW87 ashas been described. Ampicillin resistant transformants were selected onL-agar plates containing 100 mcg ml⁻¹ ampicillin.

9) Isolation of Plasmid DNA

Plasmid DNA was prepared from the colonies containing potentialhorseradish peroxidase clones essentially as described (Ish-Horowicz,D., Burke, J. F. Nucleic Acids Research 9 2989-2998 (1981).

10) Dideoxy Sequencing

The protocol used was essentially as has been described (Biggin, M. D.,Gibson, T. J., Hong, G. F. P.N.A.S. 80 3963-3965 (1983)). The method wasmodified to allow sequencing on plasmid DNA as described (Guo, L-H., Wu,R. Nucleic Acids Research 11 5521-5540 (1983).

11) Transformation

Transformation was accomplished using standard procedures. The strainused as a recipient in the cloning was HW87 which has the followinggenotype: ##STR4##

Any other standard cloning recipient such as HB101 would be adequate.

EXAMPLE 2

The front end of the synthetic HRP gene prepared in Example 1 wasmodified by the replacement of the HindIII-HpaI fragment with asynthetic linker carrying an NdeI site on the initiator ATG as follows:##STR5##

EXAMPLE 3

Expression of the Synthetic Horseradish Peroxidase Gene in Escherichiacoli

The synthetic HRP gene of Example 2 was cloned into the expressionvector pGC517 on a NdeI-BamHI fragment to give the plasmid pSD18. Thehost vector pGC517 was prepared from the known plasmid pAT153 (Twigg &Sherratt Nature 283, 216-218 (1980)), which is now a standard E. colihigh expression vector, by the incorporation by standard methods of theknown tac promoter sequence and a termination sequence. pAT153 is itselfa derivative of pBR322. In pGC517 the HRP gene is expressed from thepowerful and regulatable tac promoter. To ensure that expressionremained repressed in uninduced cultures the plasmid was maintained inE. coli strain W3110 lacI^(q), which is widely available, in which thelac repressor protein is over-produced. FIG. 5 depicts the structure ofpSD18.

Strain W3110 lacI^(q) -pSD18 was grown in M9 minimal medium containing0.2% glucose and 0.2% casamino acids. At an O.D. of 0.2-0.3 the culturewas induced by the addition of IPTG to a final concentration of 5 mM.The culture was grown for a further 3 hr with samples removed at 30 minintervals.

Microscopic examination of the induced culture revealed the presence ofinclusion bodies, characteristic of the accumulation of large amounts ofinsoluble aggregated protein within the cell. In addition, culturesexpressing HRP at high levels acquired a pink colouration, perhapsrelated to the overexpression of a haem protein. SDS/PAGE analysissubsequently revealed the presence of a large amount of a 33 kD protein,estimated at 10-20% of total cell protein in induced but not uninducedcultures. Western blot analysis confirmed that this protein was HRP.

Standard methods for inclusion body isolation could be applied to obtaina substantial purification of the denatured HRP as insoluble aggregates.This material was then dissolved in 6M guanidine HCl prior torenaturation. For renaturation, the dissolved HRP was dialysed against8M urea, 50 mM Tris HCl, 100 mM NaCl for 24 hr. Ca²⁺ was then added (asCaCl₂) to 1 mM and the sample incubated for 2 hr at room temperature.This procedure resulted in the recovery of about 0.125% of the expectedHRP activity by the standard pyrogallol colorimetric assay and based onthe protein concentration and estimated purity of the preparation (seeTable 1).

                  TABLE 1                                                         ______________________________________                                        Renaturation of HRP Expressed in E. coli                                                              Amount         Activity                                             Rate of   of recombi-                                                                           Activity                                                                             (% of max.                                           reaction  nant HRP                                                                              AU/min activity of                            Sam- Con-     (maximum) C assayed                                                                             mcg    commercial                             ple  ditions  AU/min    mcg     rec. HRP                                                                             HRP)                                   ______________________________________                                        1    before   0.01 AU/  25 mcg  5 × 10.sup.-3                                                                  0.007%                                      1st      0.8 min           AU/min                                             dialysis                   mcg                                           2    after    0.15 AU/   5.77 mcg                                                                             0.0024 0.034%                                      1st      1.1 min           AU/min                                             dialysis                   mcg                                           3    sample 2 0.01 AU/   0.76 mcg                                                                             0.029  0.125%                                      incubated                                                                              1.5 min           AU/min                                             with                       mcg                                                1 mM                                                                          Ca.sup.2+                                                                     for 2h                                                                   ______________________________________                                    

Control samples prepared from similar cultures carrying the expressionplasmid without the HRP gene gave backgrounds about 1000 fold less thanthis. The assay mixture contained freshly prepared pyrogallol andperoxide in the following concentrations: 11 mM K phosphte, pH 6.0, 8 mMH₂ O₂, 0.55% w/v pyrogallol in H₂ O. The HRP was added and the increasein adsorption at 420 nm was followed.

Thus the synthetic HRP gene is capable of high level expression in E.coli and is capable of directing the synthesis of active product.

EXAMPLE 4

The synthetic HRP gene of Example 2 was modified as follows to allow forits efficient expression in mammalian cells:

(a) The 3' end of the gene was extended from the Pst 1 site to includethe C-terminal extension reported by Fujiyama et al. Eur. J. Biochem.173, 681-687 (1988).

(b) The 5' end of the gene was modified by the addition of aHindIII/HpaI linker which encoded a signal sequence based on animmunoglobulin signalpeptide.

The modified HRP gene is depicted in FIG. 6, and will be referred to asHPRX.

EXAMPLE 5 Expression of the Synthetic Horesradish Peroxidase Gene inMammalian Cells

The HRPX gene of Example 4 was inserted into the mammlian cellexpression vector pCPH11 to give pCP21, in which the HRP gene isexpressed from the HCMV (Human Cytomegalovirus) early promoter, see FIG.7. The plasmid pCPH11 is based on pUC18, which is widely available andfrom which it can be prepared by standard methods, using the informationin FIG. 7.

The HRP expression plasmid pCP21 was transfected into COS cells usingthe standard technique of calcium phosphate precipitation (20 mcg DNAtransfected per 10⁶ cells). HRP activity was assayed in cell culturemedium, 48-72 h post transfection using tetra-methyl benzidine substrate(TMB), a standard HRP reagent. No HRP activity was detectable in controlconstructs which did not contain a signal sequence and/or the 3'extension. In contrast, HRP activity was clearly detectable in cellstransfected with pCP21 (up to 10x greater than in controls). The resultsare shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        HRP Expression in COS Cells                                                              O.D. 450                                                           Vol. Extract.                                                                            Plasmid                                                            (mcl)      pCP21     pCP22    pCP11   pCP12                                   ______________________________________                                        100        .004      .022     .008    .003                                    50         .033      .012     .010    .013                                    25         .107*     .010     .003    .016                                    10         .084*     .011     .007    .017                                     5         .064*     .015     .012    .011                                     1         .028      .008     .007    .007                                    ______________________________________                                         KEY                                                                           pCP21 HRP with N and C terminal signals, correct orientation.                 pCP22 HRP with N and C terminal signals, wrong orientation.                   pCP11 HRP with no signal sequences, correct orientation.                      pCP12 HRP with no signal sequences, wrong orientation.                        All results are the mean of duplicate samples.                                *significant level of activity.                                          

HRP Assay

For assaying cell extracts, a substrate mix was prepared as follows:

TMB (3,3',5,5' tetramethyl benzidine (Sigma)) was dissolved to 10 mg/mlin DMSO and 100 mcl of this solution added to 100 ml of assay buffer(0.1M NaAc in citric acid, pH6.0) along with 100 mcl H₂ O₂.

A cell extract was prepared by collecting the cells by centrifugationfollowed by freeze thawing or sonication. The medium, cell lysates andstandards were aliquoted in 96 well microtitre plates as follows:

    ______________________________________                                        Sample  100      50    25     10  5     1   mcl                               ______________________________________                                        Assay   0        50    75     90  95    99  mcl                               Buffer                                                                        ______________________________________                                    

Blank samples were set up using 100 mcl of assay buffer alone. 100 mclof TMB/H₂ O₂ mix was added to the samples of incubated at RT for 30 minsto 1 hour. The reaction was stopped by the addition of 50 mcl of 2.5M H₂SO₄ and the colour change read at 450 nm on a plate reader.

Commercially available HRP was used as a standard diluted by a factor of10⁻⁶.

I claim:
 1. The DNA sequence of FIG. 6, which encodes horseradishperoxidase C.
 2. A DNA construct comprising the sequence of FIG. 6 fusedto a second DNA sequence, said DNA construct encoding a hybrid proteinexhibiting horseradish peroxidase C activity.